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Cupulin is a zona pellucida-like domain protein and major component of the cupula from the inner ear.

Dernedde J, Weise C, Müller EC, Hagiwara A, Bachmann S, Suzuki M, Reutter W, Tauber R, Scherer H - PLoS ONE (2014)

Bottom Line: The extracellular membranes of the inner ear are essential constituents to maintain sensory functions, the cupula for sensing torsional movements of the head, the otoconial membrane for sensing linear movements and accelerations like gravity, and the tectorial membrane in the cochlea for hearing.So far a number of structural proteins have been described, but for the gelatinous cupula precise data are missing.Here, we describe for the first time a major proteinogenic component of the cupula structure with an apparent molecular mass of 45 kDa from salmon.

View Article: PubMed Central - PubMed

Affiliation: Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité -Universitätsmedizin Berlin, Berlin, Germany.

ABSTRACT
The extracellular membranes of the inner ear are essential constituents to maintain sensory functions, the cupula for sensing torsional movements of the head, the otoconial membrane for sensing linear movements and accelerations like gravity, and the tectorial membrane in the cochlea for hearing. So far a number of structural proteins have been described, but for the gelatinous cupula precise data are missing. Here, we describe for the first time a major proteinogenic component of the cupula structure with an apparent molecular mass of 45 kDa from salmon. Analyses of respective peptides revealed highly conserved amino-acid sequences with identity to zona pellucida-like domain proteins. Immunohistochemistry studies localized the protein in the ampulla of the inner ear from salmon and according to its anatomical appearance we identified this glycoprotein as Cupulin. Future research on structure and function of zona pellucida-like domain proteins will enhance our knowledge of inner ear diseases, like sudden loss of vestibular function and other disturbances.

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Related in: MedlinePlus

Visualization of cupula proteins.A, crude extracts from isolated cupulae from salmon, (lane 1) and chicken (lane 2) were separated on a 12% SDS-PAGE under reducing conditions and silver stained. The arrowhead highlights a dominant protein (∼45 kDa) chosen for further analyses. Lane 3, marker proteins. In the 60 kDa range additional yet unidentified protein components are visible. B, deglycosylation of salmon cupula protein extract. Lane 1, cupula extract untreated; lane 2, cupula extract+PNGase F (100 NEB units), lane 3, PNGase F control (500 NEB units). Arrowheads indicate molecular weight shift of the 45 kDa protein due to the N-deglycosylation.
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pone-0111917-g002: Visualization of cupula proteins.A, crude extracts from isolated cupulae from salmon, (lane 1) and chicken (lane 2) were separated on a 12% SDS-PAGE under reducing conditions and silver stained. The arrowhead highlights a dominant protein (∼45 kDa) chosen for further analyses. Lane 3, marker proteins. In the 60 kDa range additional yet unidentified protein components are visible. B, deglycosylation of salmon cupula protein extract. Lane 1, cupula extract untreated; lane 2, cupula extract+PNGase F (100 NEB units), lane 3, PNGase F control (500 NEB units). Arrowheads indicate molecular weight shift of the 45 kDa protein due to the N-deglycosylation.

Mentions: The cupula is a jelly-like extracellular matrix of the inner ear and part of the sensor system that measures torsional accelerations (Fig. 1). When we started to analyze the cupula protein composition from salmon and chicken by gel electrophoresis, a comparable protein pattern with ∼10–15 bands, depending on the quality of sample preparation was detected for both organisms (Fig. 2). The existence of a dominant protein that constitutes the cupula structure was predicted by Goodyear and Richardson [7]. Here we identified a prominent fuzzy band in the range of approximately 45 kDa after sample separation from salmon and chicken under denaturing and reducing conditions (Fig. 2A). Although the protein was always visible, the distinctness varied between different preparations. As extracellular matrices usually consist of glycosylated proteins we treated the cupula sample from salmon with the N-glycosidase PNGase F. Indeed the size of the dominant 45 kDa cupula protein was reduced by ∼11 kDa to a size of 34 kDa (Fig. 2 A,B). The PNGase F control (lane 3) migrated at the same position, but here we loaded the fivefold quantity of enzyme compared to the amount in lane 2 to visualize the protein. From the disappearance of the 45 kDa band and the intense staining at 34 kDa we concluded that the deglycosylated salmon protein and PNGase F run at the same position. Faintly stained protein bands in lanes 1 and 2 that migrate approximately 20 kDa below the glycosylated or deglycosylated protein, may reflect immunoreactive degradation products (Fig. 2B).


Cupulin is a zona pellucida-like domain protein and major component of the cupula from the inner ear.

Dernedde J, Weise C, Müller EC, Hagiwara A, Bachmann S, Suzuki M, Reutter W, Tauber R, Scherer H - PLoS ONE (2014)

Visualization of cupula proteins.A, crude extracts from isolated cupulae from salmon, (lane 1) and chicken (lane 2) were separated on a 12% SDS-PAGE under reducing conditions and silver stained. The arrowhead highlights a dominant protein (∼45 kDa) chosen for further analyses. Lane 3, marker proteins. In the 60 kDa range additional yet unidentified protein components are visible. B, deglycosylation of salmon cupula protein extract. Lane 1, cupula extract untreated; lane 2, cupula extract+PNGase F (100 NEB units), lane 3, PNGase F control (500 NEB units). Arrowheads indicate molecular weight shift of the 45 kDa protein due to the N-deglycosylation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219815&req=5

pone-0111917-g002: Visualization of cupula proteins.A, crude extracts from isolated cupulae from salmon, (lane 1) and chicken (lane 2) were separated on a 12% SDS-PAGE under reducing conditions and silver stained. The arrowhead highlights a dominant protein (∼45 kDa) chosen for further analyses. Lane 3, marker proteins. In the 60 kDa range additional yet unidentified protein components are visible. B, deglycosylation of salmon cupula protein extract. Lane 1, cupula extract untreated; lane 2, cupula extract+PNGase F (100 NEB units), lane 3, PNGase F control (500 NEB units). Arrowheads indicate molecular weight shift of the 45 kDa protein due to the N-deglycosylation.
Mentions: The cupula is a jelly-like extracellular matrix of the inner ear and part of the sensor system that measures torsional accelerations (Fig. 1). When we started to analyze the cupula protein composition from salmon and chicken by gel electrophoresis, a comparable protein pattern with ∼10–15 bands, depending on the quality of sample preparation was detected for both organisms (Fig. 2). The existence of a dominant protein that constitutes the cupula structure was predicted by Goodyear and Richardson [7]. Here we identified a prominent fuzzy band in the range of approximately 45 kDa after sample separation from salmon and chicken under denaturing and reducing conditions (Fig. 2A). Although the protein was always visible, the distinctness varied between different preparations. As extracellular matrices usually consist of glycosylated proteins we treated the cupula sample from salmon with the N-glycosidase PNGase F. Indeed the size of the dominant 45 kDa cupula protein was reduced by ∼11 kDa to a size of 34 kDa (Fig. 2 A,B). The PNGase F control (lane 3) migrated at the same position, but here we loaded the fivefold quantity of enzyme compared to the amount in lane 2 to visualize the protein. From the disappearance of the 45 kDa band and the intense staining at 34 kDa we concluded that the deglycosylated salmon protein and PNGase F run at the same position. Faintly stained protein bands in lanes 1 and 2 that migrate approximately 20 kDa below the glycosylated or deglycosylated protein, may reflect immunoreactive degradation products (Fig. 2B).

Bottom Line: The extracellular membranes of the inner ear are essential constituents to maintain sensory functions, the cupula for sensing torsional movements of the head, the otoconial membrane for sensing linear movements and accelerations like gravity, and the tectorial membrane in the cochlea for hearing.So far a number of structural proteins have been described, but for the gelatinous cupula precise data are missing.Here, we describe for the first time a major proteinogenic component of the cupula structure with an apparent molecular mass of 45 kDa from salmon.

View Article: PubMed Central - PubMed

Affiliation: Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité -Universitätsmedizin Berlin, Berlin, Germany.

ABSTRACT
The extracellular membranes of the inner ear are essential constituents to maintain sensory functions, the cupula for sensing torsional movements of the head, the otoconial membrane for sensing linear movements and accelerations like gravity, and the tectorial membrane in the cochlea for hearing. So far a number of structural proteins have been described, but for the gelatinous cupula precise data are missing. Here, we describe for the first time a major proteinogenic component of the cupula structure with an apparent molecular mass of 45 kDa from salmon. Analyses of respective peptides revealed highly conserved amino-acid sequences with identity to zona pellucida-like domain proteins. Immunohistochemistry studies localized the protein in the ampulla of the inner ear from salmon and according to its anatomical appearance we identified this glycoprotein as Cupulin. Future research on structure and function of zona pellucida-like domain proteins will enhance our knowledge of inner ear diseases, like sudden loss of vestibular function and other disturbances.

Show MeSH
Related in: MedlinePlus